1. Field of the Invention
The present invention relates to a method and apparatus for performing two-photon fluorescence microscopy for producing three-dimensional images of specimens.
2. Description of the Related Art
Laser fluorescence confocal microscopy is an effective technique for producing three-dimensional images. These images are created by the ability of the confocal microscope to effectively discriminate out-of-focal plane fluorescence, providing excellent depth resolution. The discrimination is achieved by placing a pinhole in front of the detector. Alternatively, two-photon absorption can be used to provide the depth discrimination.
The technique of two-photon microscopy was introduced by Denk et al. in "Two-Photon Laser Scanning Fluorescence Microscopy", Science, Vol. 248, pp. 73-76, (April, 1990). The motivation for using this method of microscopy over traditional laser fluorescence microscopy is multifold. The purported advantages include less damage to the biological system, the use of longer wavelengths which are more readily manipulated, and the ability to release caged compounds within a relatively confined vicinity.
These advantages arise from the fact that, while traditional laser fluorescence microscopy requires only a single photon .lambda..sub.1 for excitation, two-photon microscopy (as suggested by its very name) requires the simultaneous absorption of two photons .lambda..sub.2 +.lambda..sub.3 for excitation. In terms of energy, hc/.lambda..sub.1 .about.hc (1/.lambda..sub.2 +1/.lambda..sub.3). Thus, .lambda..sub.2 and .lambda..sub.3 are both longer in wavelength than .lambda..sub.1. However, it is important to note that .lambda..sub.2 need not necessarily equal .lambda..sub.3. Indeed, any combination of wavelengths can be used, as long as the net energy requirements for exciting the fluorophore are satisfied. It should also be noted that this argument can be further extended to include the simultaneous absorption of three or more wavelengths. All of these higher order, multi-photon processes result in a higher order, intensity-dependent absorption.
An example of a single-photon fluorescence microscope is illustrated in FIG. 1. Light having a wavelength .lambda..sub.1 is transmitted from light source 10 through dichroic mirror 12 and imaging lens 14 into the specimen 16 along image plane 18. The incident light excites a fluorescent medium which has been introduced into the specimen 16, and causes the fluorescent medium to emit light of a different wavelength in proportional to the average power of the incident light. The back-scattered light emitted by the fluorescent medium in the specimen 16 is reflected by the dichroic mirror 121 towards a detector 20. The same technique can be applied in a transmissive geometry as well. For the single photon case, the flux emitted by the fluorophore scales linearly with pump intensity. Thus, in a normal confocal geometry, there will be appreciable fluorescence throughout the focal volume. Hence, pinholes 22 at the conjugate image plane must be used to block the out-of-focal-plane fluorescence, in order to generate an image, as illustrated in FIG. 1.
As disclosed by Denk et al. in U.S. Pat. No. 5,034,613, a two-photon imaging system may include a laser scanning microscope, a fluorophore having the appropriate emission with long wavelength (red or infrared) illumination as a stain for a sample, a sub-picosecond laser source of appropriate wavelength, a detector for the emission of the fluorophore, and signal processing provided by a computer.
For two-photon absorption, the fluorescence signal scales as the square of the pump intensity. This intensity dependence results in appreciable fluorescence being produced only at the focus (as opposed to throughout the focal volume). Therefore, an image can, in fact, be obtained without the use of the blocking pinholes required in single photon laser fluorescence microscopy.
The squared intensity dependence dictates that the laser sources most suitable for this application operate in pulsed mode rather than in continuous wave mode. It is essential to deliver to the sample light pulses having high peak power with low total energy. The laser intensity (i.e. W/cm.sup.2) must be high enough for two photon absorption to proceed at an acceptable rate. The use of short pulses makes it possible to achieve the requisite intensities for sufficient signal-to-noise. However, above a certain energy level, pulses can cause photobleaching and possibly damage to the specimen. Accordingly, it is necessary to provide ultrashort pulse light with high peak power but low total energy. Thus, the duty-cycle of the laser is significantly reduced compared to the continuous wave excitation conditions. The reduced exposure to the pump radiation is considered a benefit to the specimen. Picosecond and femtosecond lasers are used for generating the two-photon fluorescence signal. Again, as the signal scales with the square of the intensity, most systems benefit from the considerably shorter femtosecond pulses, presently making these sources the tool of choice. The advent of reliable, solid-state femtosecond systems such as Ti:Al.sub.2 O.sub.3 and fiber lasers have also helped in this respect.
Several different sources have been used to provide ultrashort pulses to a two-photon microscope. For example, the delivery of the high peak power pulses has been made in "free space" using Ti:Sapphire and Cr:LiSAF and fiber sources. Alternatively, an optical fiber delivery system capable of compensating for dispersion introduced by optical components within the microscope can be used to delivery high peak power pulses, as disclosed by Stock et al. in U.S. application Ser. No. 08/763,381.
As reported in the above-mentioned article by Denk et al. and by Brakenhoff et al. in "Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system", Journal of Microscopy, Vol. 181, pp. 253-259 (1996), femtosecond pulses have been used successfully in taking two-photon images utilizing both point and slit excitation conditions. A point scan requires both x and y translation of the beam to create a two-dimensional field, while the slit scan requires only an x translation to create a similar two-dimensional field. Thus, higher scan rates are possible with the slit excitation condition. Three-dimensional images for either mode of excitation (point or slit) are created by measuring any number of two-dimensional data sets as a function of specimen depth. These data sets are later reconstructed in a computer to give the full three-dimensional image.
There are many different methods by which the laser beam and specimen are scanned or deflected. Acousto-optic deflectors are generally avoided in two-photon imaging systems, owing to their intrinsically large dispersion which increases the excitation pulse width and lowers the effective achievable two-photon intensity. Mirrors mounted on galvanometric scanners are the preferred method for rastering the beam, as they have negligible dispersion, and preserve the pulse width, as illustrated in FIG. 2. Several scanners must be employed to generate the sufficient degrees of freedom to map out a two-dimensional field.
In the example shown in FIG. 2, rotating mirrors 24 and 26 are rotated to scan the incident laser beam in the x and y directions in the focal plane. In order to form a three-dimensional image, the focal plane is changed by moving the sample 28 relative to the microscope objective 30 along the optical axis. A photomultiplier tube 32 can be used to detect the emission of the fluorophore transmitted through the sample 28. If a two-dimensional charge coupled device (CCD) array is used for detection (as opposed to a photomultiplier tube), the fluorescence from the sample must also be descanned in order to create the image (i.e., the florescence must be transmitted through the scanning optics in the reverse direction of the incident beam). This can further complicate the optical design of the system. Another concern with many of the scanning systems is the nonuniform scanning velocity. This could result in differences in the lateral image quality. This problem is most likely to increase with increased scan rate.
Many important biological problems would benefit from the ability to produce three-dimensional images in real-time. Current methods of acquisition and reconstruction limit the rate at which the full three-dimensional volume can be examined. More explicitly, the rate limiting factors include: the time necessary to scan either a point or line excitation source through the specimen volume under consideration, the acquisition of each two-dimensional field at a given axial depth, the transfer of this data to some form of storage media, and finally, the manipulation of these two-dimensional fields to construct a three-dimensional profile of the specimen. A secondary consideration is also the amount of information that must be maintained in the traditional two-photon laser scanning systems. For every three-dimensional image that is produced, an enormous number of data sets is generated.